The mu opiate receptor has been identified as the principal brain receptor site best correlated with the rewarding and euphoric properties of opiate drugs. Euphoric responses to rapid administration of morphine and heroin can be much more prominent than those that follow slower rates of administration. Receptor phosphorylation is thought to play a role in processes which may contribute to rate-sensitivity of opiate receptor responses, and possibly to tolerance to opiates. Phosphorylation plays important roles in modulating functional activities of numerous cellular regulatory molecules, including several G-linked receptors. Nine potential phosphoacceptor sites mutants in the human m opiate receptor were constructed during this FY and assessed for abilities to alter radiolabeled agonist and antagonist binding and/or G-coupled inhibition of adenylate cyclase. Initial observations suggest that poor levels of functional expression of mutants in serine residues in the receptor's third intracellular loop. Conceivably, these closely-spaced serines may constitute an essential component for maintaining receptor structure and/or interfacing with G proteins. In this FY, investigators in this Branch have begun to explore direct phosphorylation of the muOR. Specific anti-muOR antisera and antibodies to epitope-tagged muORs immunoprecipitated material tentatively identified as phosphor-muORs from transfected cell preparations, and m opiate receptor expression in Sf9 insect cells allowed purification of receptor protein quantities that should serve as a substrate for in vitro phosphorylation studies.